Marine Em'ironrnental Research 17 (1985) 306-309

Membrane Mimetic Electrochemistry in the Marine Sciences

A. N e l s o n

Institute for Marine Environmental Research, Natural Environment Research Council, Prospect Place, The Hoe, Plymouth, Devon PLI 3DH, Great Britain

In marine pollution studies it is generally considered that the biological availability of a particular contaminant depends to some extent on its chemical speciation, t Little is known, however, about the actual mechanism of transport of the toxic species into the organism. We consider the biomembrane transport of toxicants as the critical link between biological availability and chemical speciation. A study has thus been instigated into the transport of simple species across phospholipid monolayers as model membranes. In this paper we describe a novel technique whereby phospholipid monolayers are spread on a hanging mercury drop electrode (HMDE) and subsequently held within the bulk phase chloride electrolyte. In addition, we report preliminary elec- trochemical observations on: ( i) the form and behaviour of the monolayers in response to applied electric field and (ii) the interaction of marine pollutants, polynuclear aromatic hydrocarbons (PAHs) with the lipid fi hns.

The study of phospholipid monolayers adsorbed on mercury electrodes has been carried out previously but in these investigations the Hg adsorbed monolayer was held in equilibrium with a phospholipid layer at the air-water interface. 2'3 In our work we spread the monolayer on to mercury from the air-water interface. Subsequently the electrode is positioned within the bulk phase. Phospholipids: phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) (Lipid Products, UK) were prepared as 0.1 ~o solution in pentane. 15-20/~1 is spread on to 28 cm 2

306 Marine Environ. Res. 0141-1136/85/$03"30 ~ Elsevier Applied Science Publishers Ltd, England, 1985. Printed in Great Britain

Membrane mimetic electrochemistry in the marine sciences 307

bulk electrolyte (0.55mol/dm 3 KCI) in a thermostatted Metrohm polarographic cell. Thus the monolayer is spread in excess of 0"3 pg c m 2

which is greater than the collapse pressure, z The electrolyte is buffered at pH 8.1 with NaHCO 3 as described in earlier experiments* having been deaerated with N 2 / C O 2 mixture prior to spreading. A fresh Hg drop (0.0088 cm 2) is extruded from the capillary of a Kemula type hanging mercury drop electrode (HMDE) and mechanically lowered through the monolayer. Thus the phospholipids are transferred to the Hg surface where they remain at equilibrium spreading pressure.

A variety of lipids from different sources has been tested and it is shown that:

(i) Phospholipids with traces of impurities do not form stable monolayers.

(ii) Saturated phospholipids, e.g. dipalmitoyl PC, cannot be success- fully spread on the Hg surface.

We have found that semi-synthetic di-oleoyl PC gives the most reproducible monolayer stable for the duration of an experiment (up to 8 h). It has been established that the monolayer has fluid properties and in this respect bears similarity to natural biological membranes. Indeed, the stability is related to the fluidity of the film.

The electrochemical instrumentation and methods will be described in detail elsewhere but we perform two series of measurements. The differential capacity (Cd) of the monolayer is evaluated using out of phase (90 °) AC voltammetry and relates to the conformation and composition of the phospholipid layer. As a result capacitance measurements provide information on the penetration of the membrane by various species in solution. Metal ion transport through the film is measured using linear and square wave voltammetry.

Figure l(a) shows the Cd vs potential (U) scans of a monolayer of di- oleoyl PC. The capacitance minimum of 2/aF/cm between - 0 . 2 and - 0 . 7 V (vs Ag/AgCI 3.5 M KC1) corresponds to the hydrocarbon chains oriented adjacent to the Hg surface. The position of zero charge (PZC) of the membrane is around -0 -4 V. On either side the capacitance peaks indicate reorientation of the PC molecules. These reorientations represent membrane thinning due to an electrostriction effect. In addition, it appears that the final cathodic peak at --- - 1-25 V can be attributed to a ferroelectric switching process associated with the polar groups on the lipid molecules.

308 A. ,Velson

&O f 0

U vs Ag/~,gCt[V)

-0.5 4,2

\ ; ;,

g

Fig. I. Differential capacity (Cd) vs potential (U) for monolayers or': (a) Di-O PC; (b) Di-O PC, anthracene penetrated; (c) Di-O PC, pyrene penetrated. (b). (c) penetration

after 15 min stirring, 40,ug/litre PAH in bulk electrolyte.

The monolayer is sensitive to traces of polycyclic aromatic hydro- carbons in the bulk electrolyte phase. These moieties reversibly penetrate the uncharged membrane and alter the subsequent capacitance -potential (C d vs U) profile. In fact the capacitance minimum is lowered and the reorientation peaks are shifted in part due to a steric influence and also because of the effect of PAH penetration on the membrane electric field. Figures l(b) and (c) show the influence of PAHs anthracene and pyrene

100

% of mQx

shift of

coth 50

c~ p pe~k

o o x x

o o

x

) x

0 ' 20 ' 4'0 '

t ime ( rn ins)

Fig. 2. Di-O PC, shift of cathodic capacitance peak (','o of shift at equilibrium penetration) versus stirring time. (x) anthracene penetration; (©) phenanthrene

penetration. 40/ag/litre PAH in bulk electrolyte.

Membrane mimetic electrochemistry in the marine sciences 309

penetration on the position of the cathodic reorientation peak in the C d vs U plot. We can measure the extent of penetration by the potential shift in this peak from its initial position. The rate of penetration can also be measured as the shift of this peak, relative to the maximum shift at equilibrium, versus time (Fig. 2). When investigating isomeric hydro- carbons it is significant that initially phenanthrene penetrates the lipid layer more rapidly than anthracene.

REFERENCES

1. Nelson, A. & Donkin, P. Mar. Pollut. Bull., 16, 164--9 (1985). 2. Miller, I. R., Rishpon, J. & Tenenbaum,'A. Bioelectrochem. Bioenerg., 1,

528-42 (1976). 3. Lecompte, M. F. & Miller, I. R. Biochem., 19, 3439--46 (1980). 4. Nelson, A. & Mantoura, R. F. C. J. Electroanal. Chem., 164, 237-52 (1984).